Joo Seok Han

Interaction of SeqA and Dam Methylase on the Hemimethylated Origin of Escherichia coli Chromosomal DNA Replication

Preferential binding of SeqA protein to hemimethylated oriC, the origin of Escherichia coli chromosomal replication, delays methylation by Dam methylase. Because the SeqA-oriC interaction appears to be essential in timing of chromosomal replication initiation, the biochemical functions of SeqA protein and Dam methylase at the 13-mer L, M, and R region containing 4 GATC sequences at the left end oforiC were examined. We found that SeqA protein preferentially bound hemimethylated 13-mers but not fully nor unmethylated 13-mers. Regardless of strand methylation, the binding of SeqA protein to the hemimethylated GATC sequence of 13-mer L was followed by additional binding to other hemimethylated GATC sequences of 13-mer M and R. On the other hand, Dam methylase did not discriminate binding of 13-mers in different methylation patterns and was not specific to GATC sequences. The binding specificity and higher affinity of SeqA protein over Dam methylase to the hemimethylated 13-mers along with the reported cellular abundance of this protein explains the dominant action of SeqA protein over Dam methylase to the newly replicated oriC for the sequestration of chromosomal replication. Furthermore, SeqA protein bound to hemimethylated 13-mers was not dissociated by Dam methylase, and most SeqA protein spontaneously dissociated 10 min after binding. Also, SeqA protein delayed thein vitro methylation of hemimethylated 13-mers by Dam methylase. These in vitro results suggest that the intrinsic binding instability of SeqA protein results in release of sequestrated hemimethylated oriC.

Dimeric configuration of SeqA protein bound to a pair of hemi-methylated GATC sequences

The binding of SeqA protein to hemi-methylated GATC sequences (hemi-sites) regulates chromosome initiation and the segregation of replicated chromosome in Escherichia coli. We have used atomic force microscopy to examine the architecture of SeqA and the mode of binding of one molecule of SeqA to a pair of hemi-sites in aqueous solution. SeqA has a bipartite structure composed of a large and a small lobe. Upon binding of a SeqA molecule to a pair of hemi-sites, the larger lobe becomes visibly separated into two DNA binding domains, each of which binds to one hemi-site. The two DNA binding domains are held together by association between the two multimerization domains that make up the smaller lobe. The binding of each DNA binding domain to a hemi-site leads to bending of the bound DNA inwards toward the bound protein. In this way, SeqA adopts a dimeric configuration when bound to a pair of hemi-sites. Mutational analysis of the multimerization domain indicates that, in addition to multimerization of SeqA polypeptides, this domain contributes to the ability of SeqA to bind to a pair of hemi-sites and to its cooperative behavior.

Trigger factor interacts with DnaA protein to stimulate its interaction with DnaA box

While screening proteins that interact with DnaA protein, the initiator protein for Escherichia coli chromosomal DNA replication, we found a 52‐kD sized protein which bound to DnaA protein in a salt‐dependent manner. This protein was identified as trigger factor, a ribosome‐associated peptidyl‐prolyl‐cis/trans isomerase with chaperone activity. Trigger factor was overproduced and purified to near homogeneity, and its effect on the function of DnaA protein was examined. Enhanced binding of DnaA protein to DnaA box with no apparent super shift in the gel‐shift experiments suggested that trigger factor, by virtue of its chaperone activity, exerts a change on DnaA protein thus increasing its binding affinity for DnaA box.

Proteolysis of the reveise transcriptase of hepatitis B virus by ion protease in E coli

Hepatitis B virus (HBV) polymerase, which possesses the activities of terminal binding, DNA polymerase, reverse transcriptase and RNaseH, has been shown to accomplish viral DNA replication through a pregenomic intermediate. Because the HBV polymerase has not been purified, the expression of HBV polymerase was examined in an E. coli expression system that is under the regulation of arabinose operon. The expressed individual domain containing terminal binding protein, polymerase, or RNaseH turned out to be insoluble. The activities of those domains were not able to be recovered by denaturation and renaturation using urea or guanidine‐HCl. The expressed reverse transcriptase containing the polymerase and RNaseH domains became extensively degraded, whereas the proteolysis was reduced in a lon‐ mutant. These results indicate that Lon protease proteolyzes the HBV reverse transcriptase expressed in E. coli.